These studies should enhance our understanding of the molecular basis for human alpha and beta chorionic gonadotropin (hCG) gene expression in normal and neoplastic tissues. This laboratory will extend the 5'-length of the cloned alpha and beta hCG cDNAs already available. The chromosomal location of human CG genes will be determined using somatic cell hybrids of known chromosomal content for assignment of hCG genes to specific chromosomes, together with Southern blottingof the hybrid cell DNA and labeled hCG cDNA probes. Natural alpha and beta hCG genes will be selected from human genomic libraries; their structures will be compared to those from normal placentae and trophoblastic and nontrophoblastic neoplasms to evaluate gene number and possible rearrangements, again by Southern blotting. Chromatin structure of hCG genes, as reflected by DNase sensitivity and methylation, will be elucidated in the various normal and neoplastic tissues. Specific sequences in the 5'-flanking region of alpha and beta hCG genes that may act as promoters of gene expression will be defined by testing specific deletion mutants of natural hCG genes in a cell-free transcription system. To study the nature of the mRNAs transcribed from alpha and beta CG genes in normal placentae, malignant trophoblastic cell lines, and malignant tumors secreting hCG or one of its subunits, we will determine the processing of nuclear precursors to mature cytoplasmic hCG mRNAs by the Northern blotting method. The effects of apparent stimulators of hCG production such as epidermal growth factor, sodium butyrate, and dibutyryl cAMP, will be investigated. The molecular level of the regulation will be elucidated: transcription initiation using isolated nuclei, mRNA processing (levels of nuclear and cytoplasmic mRNA), and translational efficiency as determined in the reticulocyte lysate translation system, as well as post-translational effects on alpha and hCG-beta protein. Lastly, human alpha and beta hCG chromosomal genes will be transferred into eukaryotic cells not expressing these genes to see whether expression will be obtained, including proper subunit processing and combination, when both genes are simultaneously introduced. Specific hCG gene deletion mutants will also be used to evaluate effects on subsequent expression. These studies should provide insights into the association of hCG production and neoplastic transformation, as well as increase our understanding of normal alpha and beta hCG gene expression. (C)